A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

Document Type: Original Article

Authors

1 Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

2 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

3 Institute of Human Genetics, North East England, Stem Cell Institute, International Center for Life, Newcastle University, Newcastle, UK

4 Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran

5 Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6 Magee-Womens Research Institute & Foundation, University of Pittsburgh Medical Sciences, Pittsburgh, PA 15213, USA

7 Department of Genetics, Faculty of Sciences, Islamic Azad University, Rasht Branch, Rasht, Iran

Abstract

Objective(s):To culture thein vitro mouse embryonic stem cells (mESCs) and to direct their  differentiation to germ-line cells; in present study we used a vector backbone containing the fusion construct Stra8-EGFP to select differentiated ES cells that entered meiosis.  Retinoic acid was used to differentiate embryonic stem cells to germ cells.
Materials and Methods: A fragment of Stra8 gene promoter (-1400 to +7) was inserted in ScaI/HindIII multiple cloning site of pEGFP-1 vector. Theelectroporationwas done on embryonic stem cells and positive colonies were selected as puromycin-resistant after three weeks of treatment with puromycin. All-trans retinoic acid (RA) was used for differentiation of mESCs at final concentration of 10-5M. The expression of protamine 1 (Prm1) gene was checked as post meiotic marker in differentiated mESCs after 5, 10, 15, 21 and 30 days after RA induction.
Results: The PCR amplification by specific primers for Stra8-EGFP fusion gene was detected in DNA sample from mESCs after electroporation and puromycin treatment.  GFP-positive mESC colonies were observed after 72 hr RA induction. The protamine 1 gene was expressed after 21 days of RA induction.
Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

Keywords


1.Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula M, et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development 1990; 110:815-821.

2.Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292:154-156.

3.Smith A. A glossary for stem-cell biology. Nature 2006; 441:1060.

4.Rathjen PD, Toth S, Willis A, Heath JK, Smith AG. Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell 1990; 62:1105-1114.

5.Oulad-Abdelghani M, Bouillet P, Decimo D, Gansmuller A, Heyberger S, Dolle P, et al. Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene. J Cell Biol 1996; 135:469-477.

6.Nayernia K, Li M, Jaroszynski L, Khusainov R, Wulf G, Schwandt I, et al. Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells. Hum Mol Genet 2004; 13:1451-1460.

7.Joyner AL. Gene Targeting: A Practical Approach. 2nd ed. Oxford: Oxford University Press; 2000.

8.Miryounesi M, Nayernia K, Dianatpour M, Mansouri F, Modarressi MH. Co-culture of mouse embryonic stem cells with sertoli cells promote in vitro generation of germ cells. Iran J Basic Med Sci 2013; 16:779-783.

9.Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300:1251-1256.

10.Sato T, Katagiri K, Yokonishi T, Kubota Y, Inoue K, Ogonuki N, et al. In vitro production of fertile sperm from murine spermatogonial stem cell lines. Nat Commun 2011; 2:472.

11.Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003; 100:11457-11462.

12.Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004; 427:148-154.

13.Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981; 78:7634-7638.

14.Rathjen PD, Lake J, Whyatt LM, Bettess MD, Rathjen J. Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. Reprod Fertil Dev 1998; 10:31-47.

15.Aflatoonian B, Ruban L, Jones M, Aflatoonian R, Fazeli A, Moore HD. In vitro post-meiotic germ cell development from human embryonic stem cells. Hum Reprod 2009; 24:3150-3159.

16.West FD, Machacek DW, Boyd NL, Pandiyan K, Robbins KR, Stice SL. Enrichment and differentiation of human germ-like cells mediated by feeder cells and basic fibroblast growth factor signaling. Stem Cells 2008; 26:2768-2776.

17.Nayernia K, Nolte J, Michelmann HW, Lee JH, Rathsack K, Drusenheimer N, et al. In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 2006; 11:125-132.

18.Chen W, Jia W, Wang K, Zhou Q, Leng Y, Duan T, et al. Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway. Biochem Biophys Res Commun 2012; 418:571-577.

19.Miyamoto T, Sengoku K, Takuma N, Hasuike S, Hayashi H, Yamauchi T, et al. Isolation and expression analysis of the testis-specific gene, STRA8, stimulated by retinoic acid gene 8. J Assist Reprod Genet 2002; 19:531-535.

20.Baltus AE, Menke DB, Hu YC, Goodheart ML, Carpenter AE, de Rooij DG, et al. In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication. Nat Genet 2006; 38:1430-1434.

21.Lu XY, Yang B, Xu SF, Zou T. STRA8 as a specific expression marker in postnatal male germ cells. Zhonghua Nan Ke Xue 2010; 16:161-165.

22.Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC. Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proc Natl Acad Sci USA 2006; 103:2474-2479.